Bispecific antigen binding proteins

ABSTRACT

The present invention relates to bispecific antigen binding proteins, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/495,641, filed Apr. 24, 2017, now U.S. Pat. No. 10,640,555, issuedMay 5, 2020, which is a divisional of U.S. application Ser. No.12/780,971 filed May 17, 2010, now U.S. Pat. No. 9,676,845, issued Jun.13, 2017, which claims the benefit of European Patent Application No.09007857 filed, Jun. 16, 2009, each of which is hereby incorporated byreference in its entirety.

The present invention relates to bispecific antigen binding proteins,methods for their production, pharmaceutical compositions containingsaid protein, and uses thereof.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392020201SEQLIST.TXT,date recorded: Apr. 2, 2020, size: 26 KB).

BACKGROUND OF THE INVENTION

Engineered proteins, such as bi- or multispecific antibodies capable ofbinding two or more antigens, are known in the art. Such multispecificbinding proteins can be generated using cell fusion, chemicalconjugation, or recombinant DNA techniques.

A wide variety of recombinant multispecific antibody formats have beendeveloped in the recent past, e.g. tetravalent bispecific antibodies byfusion of, e.g. an IgG antibody format and single chain domains (seee.g. Coloma, M. J., et. al., Nature Biotech. 15 (1997) 159-163; WO2001/077342; and Morrison, S. L., Nature Biotech. 25 (2007) 1233-1234.

Also several other new formats wherein the antibody core structure (IgA,IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- ortetrabodies, minibodies, several single chain formats (scFv, Bis-scFv),which are capable of binding two or more antigens, have been developed(Holliger, P., et. al., Nature Biotech 23 (2005) 1126-1136; Fischer, N.,and Léger, O., Pathobiology 74 (2007) 3-14; Shen, J., et. al., J.Immunol. Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech 25(2007) 1290-1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD,IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fusee.g. two Fab fragments or scFv (Fischer, N., and Léger, O., Pathobiology74 (2007) 3-14). While it is obvious that linkers have advantages forthe engineering of bispecific antibodies, they may also cause problemsin therapeutic settings. Indeed, these foreign peptides might elicit animmune response against the linker itself or the junction between theprotein and the linker. Further more, the flexible nature of thesepeptides makes them more prone to proteolytic cleavage, potentiallyleading to poor antibody stability, aggregation and increasedimmunogenicity. In addition one may want to retain effector functions,such as e.g. complement-dependent cytotoxicity (CDC) or antibodydependent cellular cytotoxicity (ADCC), which are mediated through theFc part by maintaining a high degree of similarity to naturallyoccurring antibodies.

Thus ideally, one should aim at developing bispecific antibodies thatare very similar in general structure to naturally occurring antibodies(like IgA, IgD, IgE, IgG or IgM) with minimal deviation from humansequences.

In one approach bispecific antibodies that are very similar to naturalantibodies have been produced using the quadroma technology (seeMilstein, C. and Cuello, A. C., Nature 305 (1983) 537-540) based on thesomatic fusion of two different hybridoma cell lines expressing murinemonoclonal antibodies with the desired specificities of the bispecificantibody. Because of the random pairing of two different antibody heavyand light chains within the resulting hybrid-hybridoma (or quadroma)cell line, up to ten different antibody species are generated of whichonly one is the desired, functional bispecific antibody. Due to thepresence of mispaired byproducts, and significantly reduced productionyields, means sophisticated purification procedures are required (seee.g. Morrison, S. L., Nature Biotech. 25 (2007) 1233-1234). In generalthe same problem of mispaired byproducts remains if recombinantexpression techniques are used.

An approach to circumvent the problem of mispaired byproducts, which isknown as ‘knobs-into-holes’, aims at forcing the pairing of twodifferent antibody heavy chains by introducing mutations into the CH3domains to modify the contact interface. On one chain bulky amino acidswere replaced by amino acids with short side chains to create a ‘hole’.Conversely, amino acids with large side chains were introduced into theother CH3 domain, to create a ‘knob’. By coexpressing these two heavychains (and two identical light chains, which have to be appropriate forboth heavy chains), high yields of heterodimer formation (‘knob-hole’)versus homodimer formation (‘hole-hole’ or ‘knob-knob’) was observed(Ridgway, J. B., Protein Eng. 9 (1996) 617-621; and WO 96/027011). Thepercentage of heterodimer could be further increased by remodeling theinteraction surfaces of the two CH3 domains using a phage displayapproach and the introduction of a disulfide bridge to stabilize theheterodimers (Merchant A. M, et al., Nature Biotech 16 (1998) 677-681;Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35). New approaches forthe knobs-into-holes technology are described in e.g. in EP 1870459A1.Although this format appears very attractive, no data describingprogression towards the clinic are currently available. One importantconstraint of this strategy is that the light chains of the two parentantibodies have to be identical to prevent mispairing and formation ofinactive molecules. Thus this technique is not appropriate for easilydeveloping recombinant, bispecific antibodies against two antigensstarting from two antibodies against the first and the second antigen,as either the heavy chains of these antibodies an/or the identical lightchains have to be optimized.

Another approach to circumvent the problem of mispaired byproducts inthe preparation of bispecific antibodies, is to switch from heterodimersto homodimers by using an full length antibody which specifically bindsto a first antigen and which has fused to its heavy chains N-termini twofused Fab fragments which specifically bind to a second antigen asdescribed e.g. in WO2001/077342. One important disadvantage of thisstrategy is the formation of undesired inactive byproducts by themispairing of the light chains of the full length antibody with theCH1-VH domains of the Fab fragments and by the mispairing of the Fabfragment light chains with CH1-VH domains of the full length antibody.

WO 2006/093794 relates to heterodimeric protein binding compositions. WO99/37791 describes multipurpose antibody derivatives. Morrison, S., L.,et al the J. Immunolog, 160(1998) 2802-2808 refers to the influence ofvariable region domain exchange on the functional properties of IgG.

SUMMARY OF THE INVENTION

The invention comprises a bispecific antigen binding protein,comprising:

-   -   a) two light chains and two heavy chains of an antibody that        comprises two Fab fragments and that specifically binds to a        first antigen; and    -   b) two additional Fab fragments of an antibody which        specifically binds to a second antigen, wherein the additional        Fab fragments are both fused via a peptide connector either at        the C- or N-termini of the heavy chains of a);        -   wherein the bispecific antigen binding protein also            comprises a structural modification selected from the group            consisting of:            -   i) in both Fab fragments of a) or in both Fab fragments                of b) the variable domains VL and VH are replaced by                each other, and the constant domains CL and CH1 are                replaced by each other, or the constant domains CL and                CH1 are replaced by each other;            -   ii) in both Fab fragments of a)            -   the variable domains VL and VH are replaced by each                other,            -   and            -   the constant domains CL and CH1 are replaced by each                other, and in both Fab fragments of b)            -   the variable domains VL and VH are replaced by each                other,            -   or            -   the constant domains CL and CH1 are replaced by each                other;            -   iii) in both Fab fragments of a)            -   the variable domains VL and VH are replaced by each                other,            -   or            -   the constant domains CL and CH1 are replaced by each                other, and in both Fab fragments of b)            -   the variable domains VL and VH are replaced by each                other,            -   and            -   the constant domains CL and CH1 are replaced by each                other;            -   v) in both Fab fragments of a)            -   the variable domains VL and VH are replaced by each                other, and            -   in both Fab fragments of b)            -   the constant domains CL and CH1 are replaced by each                other; and            -   v) in both Fab fragments of a)            -   the constant domains CL and CH1 are replaced by each                other, and            -   in both Fab fragments of b)            -   the variable domains VL and VH are replaced by each                other.

A further embodiment of the invention is a method for the preparation ofan antigen binding protein according to the invention comprising thesteps of

-   -   a) transforming a host cell with        -   vectors comprising nucleic acid molecules encoding a            bispecific antigen binding protein according to the            invention    -   b) culturing the host cell under conditions that allow synthesis        of said antibody molecule; and    -   c) recovering said antibody molecule from said culture.

A further embodiment of the invention is a host cell comprising

-   -   vectors comprising nucleic acid molecules encoding an antigen        binding protein according to the invention

A further embodiment of the invention is a pharmaceutical compositioncomprising an antigen binding protein according to the invention and atleast one pharmaceutically acceptable excipient.

A further embodiment of the invention is a method for the treatment of apatient in need of therapy, comprising administering to the patient atherapeutically effective amount of an antigen binding protein accordingto the invention.

According to the invention, the ratio of a desired bispecific antigenbinding protein compared to undesired side products can be improved bythe replacement of certain domains a) in the Fab fragment of the fulllength antibody which specifically binds to the a first antigen and/orb) in the two additional fused Fab fragments. In this way the undesiredmispairing of the light chains with the wrong CH1-VH domains can bereduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic structure of a full length antibody without CH4 domainspecifically binding to a first antigen 1 with two pairs of heavy andlight chain which comprise variable and constant domains in a typicalorder.

FIGS. 2a and 2b Schematic structure of typical unmodified Fab fragmentsspecifically binding to a second antigen 2 with the peptide connectoreither at the C-terminus (FIG. 2a ) or N-terminus (FIG. 2b ) of theCH1-VH chain.

FIGS. 3a, 3b and 3c Schematic structure of a full length antibodyspecifically binding to a first antigen 1 which has fused to theN-terminus of its heavy chain two unmodified Fab fragments specificallybinding to a second antigen 2 (FIG. 3a ) and the undesired side productsdue to mispairing (FIG. 3b and FIG. 3c ).

FIGS. 4a, 4b and 4c Schematic structure of a bispecific antigen bindingproteins according to the invention in which the mispairing is reducedby the replacement of certain domains a) in the Fab fragment of the fulllength antibody which specifically binds to a first antigen 1 and/or b)in the two additional fused Fab fragments antibody which specificallybinds to a second antigen 2. FIG. 4a shows bispecific antigen bindingproteins. FIGS. 4b and 4c show all combination of VH/VL and/or CH1/CLdomain exchanges within the full length Fab fragments and the additionalFab fragments which lead to bispecific antigen binding proteinsaccording to the invention with reduced mispairing.

FIG. 5 Schematic structure of a bispecific antigen binding proteinsaccording to the invention recognizing Ang-2 and VEGF (Example 1).

FIG. 6 Schematic structure of a bispecific antigen binding proteinsaccording to the invention recognizing Ang-2 and VEGF (Example 2).

FIG. 7 Schematic structure of a bispecific antigen binding proteinsaccording to the invention recognizing Ang-2 and VEGF (Example 3).

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises a bispecific antigen binding protein,comprising:

-   -   a) two light chains and two heavy chains of an antibody that        comprises two Fab fragments and that specifically binds to a        first antigen; and    -   b) two additional Fab fragments of an antibody which        specifically binds to a second antigen, wherein the additional        Fab fragments are both fused via a peptide connector either at        the C- or N-termini of the heavy chains of a);        -   wherein the bispecific antigen binding protein also            comprises a structural modification selected from the group            consisting of:            -   i) in both Fab fragments of a) or in both Fab fragments                of b)            -   the variable domains VL and VH are replaced by each                other,            -   and the constant domains CL and CH1 are replaced by each                other, or the constant domains CL and CH1 are replaced                by each other;            -   ii) in both Fab fragments of a)            -   the variable domains VL and VH are replaced by each                other,            -   and            -   the constant domains CL and CH1 are replaced by each                other, and in both Fab fragments of b)            -   the variable domains VL and VH are replaced by each                other,            -   or            -   the constant domains CL and CH1 are replaced by each                other;            -   iii) in both Fab fragments of a)            -   the variable domains VL and VH are replaced by each                other,            -   or            -   the constant domains CL and CH1 are replaced by each                other, and in both Fab fragments of b)            -   the variable domains VL and VH are replaced by each                other,            -   and            -   the constant domains CL and CH1 are replaced by each                other;            -   v) in both Fab fragments of a)            -   the variable domains VL and VH are replaced by each                other, and            -   in both Fab fragments of b)            -   the constant domains CL and CH1 are replaced by each                other; and            -   v) in both Fab fragments of a)            -   the constant domains CL and CH1 are replaced by each                other, and in both Fab fragments of b)            -   the variable domains VL and VH are replaced by each                other.

One embodiment of the invention is the bispecific antigen bindingprotein according to the invention wherein

-   -   the additional Fab fragments are fused both via a peptide        connector either to the C-termini of the heavy chains of a), or        to the N-termini of the heavy chains of a).

Another embodiment of the invention is the bispecific antigen bindingprotein according to the invention wherein

-   -   the additional Fab fragments are fused both via a peptide        connector either to the C-termini of the heavy chains of a).

Another embodiment of the invention is the bispecific antigen bindingprotein according to the invention wherein

-   -   the additional Fab fragments are fused both via a peptide        connector to the N-termini of the heavy chains of a).

Another embodiment of the invention is the bispecific antigen bindingprotein according to the invention wherein the bispecific antigenbinding protein also comprises a structural modification selected fromthe group consisting of:

-   -   i) in both Fab fragments of a), or in both Fab fragments of b),        the variable domains VL and VH are replaced by each other,        and/or    -   the constant domains CL and CH1 are replaced by each other.

Another embodiment of the invention is the bispecific antigen bindingprotein according to the invention comprising the following structuralmodifications:

-   -   i) in both Fab fragments of a)    -   the variable domains VL and VH are replaced by each other,        and/or    -   the constant domains CL and CH1 are replaced by each other.

Another embodiment of the invention is the bispecific antigen bindingprotein according to the invention comprising the following structuralmodifications:

-   -   i) in both Fab fragments of a)    -   the constant domains CL and CH1 are replaced by each other.

Another embodiment of the invention is the bispecific antigen bindingprotein according to the invention comprising the following structuralmodifications:

-   -   i) in both Fab fragments of b)    -   the variable domains VL and VH are replaced by each other,        and/or    -   the constant domains CL and CH1 are replaced by each other.

Another embodiment of the invention is the bispecific antigen bindingprotein according to the invention comprising the following structuralmodifications

-   -   i) in both Fab fragments of b)    -   the constant domains CL and CH1 are replaced by each other.

According to the invention, the ratio of a desired bispecific antigenbinding protein compared to undesired side products (due to mispairingof the light chains with the wrong CH1-VH domains) can be reduced by thereplacement of certain domains a) in the Fab fragment of the full lengthantibody which specifically binds to the a first antigen and/or b) inthe two additional fused Fab fragments. Mispairing in this connectionmeans the association of i) the light chain of the full length antibodyunder a) with CH1-VH domains of the Fab fragments under b); or ii) thelight chain of the Fab fragments under b) with the CH1-VH domains of thefull length antibody under a) (see FIG. 3) which leads to undesiredinactive or not fully functional byproducts.

The term “antibody” as used herein denotes a full length antibodyconsisting of two antibody heavy chains and two antibody light chains(see FIG. 1). A heavy chain of full length antibody is a polypeptideconsisting in N-terminal to C-terminal direction of an antibody heavychain variable domain (VH), an antibody constant heavy chain domain 1(CH1), an antibody hinge region (HR), an antibody heavy chain constantdomain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3),abbreviated as VH-CH1-HR-CH2-CH3; and optionally an antibody heavy chainconstant domain 4 (CH4) in case of an antibody of the subclass IgE.Preferably the heavy chain of full length antibody is a polypeptideconsisting in N-terminal to C-terminal direction of VH, CH1, HR, CH2 andCH3. The light chain of full length antibody is a polypeptide consistingin N-terminal to C-terminal direction of an antibody light chainvariable domain (VL), and an antibody light chain constant domain (CL),abbreviated as VL-CL. The antibody light chain constant domain (CL) canbe K (kappa) or λ, (lambda). The antibody chains are linked together viainter-polypeptide disulfide bonds between the CL domain and the CH1domain (i.e. between the light and heavy chain) and between the hingeregions of the full length antibody heavy chains. Examples of typicalfull length antibodies are natural antibodies like IgG (e.g. IgG 1 andIgG2), IgM, IgA, IgD, and IgE.) The antibodies according to theinvention can be from a single species e.g. human, or they can bechimerized or humanized antibodies. The full length antibodies accordingto the invention comprise two antigen binding sites each formed by apair of VH and VL, which both specifically bind to the same (first)antigen. The C-terminus of the heavy or light chain of the full lengthantibody denotes the last amino acid at the C-terminus of the heavy orlight chain. The antibody comprises two identical Fab fragmentsconsisting of the VH and CH1 domain of the heavy chain and the VL and CLdomain of the light chain. (see FIGS. 1 and 2).

An “additional Fab fragment” (see FIG. 2) of an antibody whichspecifically binds to a second antigen refers to a further Fab fragmentconsisting of the VH and CH1 domain of the heavy chain and the VL and CLdomain of the light chain of the second antibody. The additional Fabfragments are fused in their unmodified form (see FIG. 3) via the heavychain part (either CH1 or VH domain) to the C- or N-termini of the heavychains or light chains of the antibody specifically binding to a firstantigen.

The term “peptide connector” as used within the invention denotes apeptide with amino acid sequences, which is preferably of syntheticorigin. These peptide connectors according to invention are used to fusethe antigen binding peptides to the C- or N-terminus of the full lengthand/or modified full length antibody chains to form a bispecific antigenbinding protein according to the invention. Preferably the peptideconnectors under c) are peptides with an amino acid sequence with alength of at least 5 amino acids, preferably with a length of 5 to 100,more preferably of 10 to 50 amino acids. In one embodiment the peptideconnector is (GxS)n or (GxS)nGm with G=glycine, S=serine, and (x=3, n=3,4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or3), preferably x=4 and n=2 or 3, more preferably with x=4, n=2. In oneembodiment the peptide connector is (G₄S)₂.

The terms “binding site” or “antigen-binding site” as used hereindenotes the region(s) of an antigen binding protein according to theinvention to which a ligand (e.g. the antigen or antigen fragment of it)actually binds and which is derived from an antibody molecule or afragment thereof (e.g. a Fab fragment). The antigen-binding siteaccording to the invention comprises an antibody heavy chain variabledomains (VH) and an antibody light chain variable domains (VL).

The antigen-binding sites (i. the pairs of VH/VL) that specifically bindto the desired antigen can be derived a) from known antibodies to theantigen or b) from new antibodies or antibody fragments obtained by denovo immunization methods using inter alia either the antigen protein ornucleic acid or fragments thereof or by phage display.

An antigen-binding site of an antigen binding protein of the inventioncontains six complementarity determining regions (CDRs) which contributein varying degrees to the affinity of the binding site for antigen.There are three heavy chain variable domain CDRs (CDRH1, CDRH2 andCDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 andCDRL3). The extent of CDR and framework regions (FRs) is determined bycomparison to a compiled database of amino acid sequences in which thoseregions have been defined according to variability among the sequences.

Antibody specificity refers to selective recognition of the antibody orantigen binding protein for a particular epitope of an antigen. Naturalantibodies, for example, are monospecific. Bispecific antibodies areantibodies which have two different antigen-binding specificities. Wherean antibody has more than one specificity, the recognized epitopes maybe associated with a single antigen or with more than one antigen.

The term “monospecific” antibody or antigen binding protein as usedherein denotes an antibody or antigen binding protein that has one ormore binding sites each of which bind to the same epitope of the sameantigen.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in an antibody molecule.A natural antibody for example or a full length antibody according tothe invention has two binding sites and is bivalent. The term“tetravalent”, denotes the presence of four binding sites in an antigenbinding protein. The term “tetravalent, bispecific” as used hereindenotes antigen binding protein according to the invention that has fourantigen-binding sites of which two binds to another antigen (or anotherepitope of the antigen). Antigen binding proteins of the presentinvention have four binding sites and are tetravalent.

The full length antibodies of the invention comprise immunoglobulinconstant regions of one or more immunoglobulin classes Immunoglobulinclasses include IgG, IgM, IgA, IgD, and IgE isotypes and, in the case ofIgG and IgA, their subtypes. In a preferred embodiment, an full lengthantibody of the invention has a constant domain structure of an IgG typeantibody.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody or antibody or antigenbinding protein molecules of a single amino acid composition.

The term “chimeric antibody” refers to an antibody comprising a variableregion, i.e., binding region, from one source or species and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a murine variable region and a human constant region arepreferred. Other preferred forms of “chimeric antibodies” encompassed bythe present invention are those in which the constant region has beenmodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding. Such chimeric antibodies arealso referred to as “class-switched antibodies.”. Chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding immunoglobulin variable regions and DNA segmentsencoding immunoglobulin constant regions. Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques are well known in the art. See, e.g., Morrison, S., L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos.5,202,238 and 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric antibodies. Other forms of “humanizedantibodies” encompassed by the present invention are those in which theconstant region has been additionally modified or changed from that ofthe original antibody to generate the properties according to theinvention, especially in regard to C1q binding and/or Fc receptor (FcR)binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;Brueggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodiescan also be produced in phage display libraries (Hoogenboom, H. R., andWinter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J.Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. andBoerner et al. are also available for the preparation of humanmonoclonal antibodies (Cole, S., P., C., et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss (1985) 77-96; and Boerner, P., et al.,J. Immunol. 147 (1991) 86-95). As already mentioned for chimeric andhumanized antibodies according to the invention the term “humanantibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to C1q binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation.)

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The “variable domain” (variable domain of a light chain (VL), variableregion of a heavy chain (VH) as used herein denotes each of the pair oflight and heavy chains which is involved directly in binding theantibody to the antigen. The domains of variable human light and heavychains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementarity determiningregions, CDRs). The framework regions adopt a β-sheet conformation andthe CDRs may form loops connecting the β-sheet structure. The CDRs ineach chain are held in their three-dimensional structure by theframework regions and form together with the CDRs from the other chainthe antigen binding site. The antibody heavy and light chain CDR3regions play a particularly important role in the bindingspecificity/affinity of the antibodies according to the invention andtherefore provide a further object of the invention.

The terms “hypervariable region” or “antigen-binding portion of anantibody” when used herein refer to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. CDRs on each chain are separated by such framework aminoacids. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding. CDR and FR regions are determinedaccording to the standard definition of Kabat, et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991).

As used herein, the term “binding” or “specifically binding” refers tothe binding of the antibody to an epitope of the antigen in an in vitroassay, preferably in an plasmon resonance assay (BIAcore, GE-HealthcareUppsala, Sweden) with purified wild-type antigen. The affinity of thebinding is defined by the terms ka (rate constant for the association ofthe antibody (or antibody or antigen binding protein) from theantibody/antigen complex), k_(D) (dissociation constant), and K_(D)(k_(D)/ka). Binding or specifically binding means a binding affinity(K_(D)) of 10⁻⁸ mol/l or less, preferably 10⁻⁹ M to 10⁻¹³ mol/l. Thus, abispecific antigen binding protein according to the invention isspecifically binding to each antigen for which it is specific with abinding affinity (K_(D)) of 10⁻⁸ mol/l or less, preferably 10⁻⁹ M to10⁻¹³ mol/l.

Binding of the antibody to the FcγRIII can be investigated by a BIAcoreassay (GE-Healthcare Uppsala, Sweden). The affinity of the binding isdefined by the terms ka (rate constant for the association of theantibody from the antibody/antigen complex), k_(D) (dissociationconstant), and K_(D) (k_(D)/ka).

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody.

In certain embodiments, an antibody is the to specifically bind anantigen when it preferentially recognizes its target antigen in acomplex mixture of proteins and/or macromolecules.

In a further embodiment the bispecific antigen binding protein accordingto the invention is characterized in that the full length antibody is ofhuman IgG1 subclass, or of human IgG1 subclass with the mutations L234Aand L235A.

In a further embodiment the bispecific antigen binding protein accordingto the invention is characterized in that the full length antibody is ofhuman IgG2 subclass.

In a further embodiment the bispecific antigen binding protein accordingto the invention is characterized in that the full length antibody is ofhuman IgG3 subclass.

In a further embodiment the bispecific antigen binding protein accordingto the invention is characterized in that the full length antibody is ofhuman IgG4 subclass or, of human IgG4 subclass with the additionalmutation S228P.

Preferably the bispecific antigen binding protein according to theinvention is characterized in that the full length antibody is of humanIgG1 subclass, of human IgG4 subclass with the additional mutationS228P.

It has now been found that the bispecific antigen binding proteinsaccording to the invention have improved characteristics such asbiological or pharmacological activity, pharmacokinetic properties ortoxicity. They can be used e.g. for the treatment of diseases such ascancer.

The term “constant region” as used within the current applicationsdenotes the sum of the domains of an antibody other than the variableregion. The constant region is not involved directly in binding of anantigen, but exhibits various effector functions. Depending on the aminoacid sequence of the constant region of their heavy chains, antibodiesare divided in the classes: IgA, IgD, IgE, IgG and IgM, and several ofthese may be further divided into subclasses, such as IgG1, IgG2, IgG3,and IgG4, IgA1 and IgA2. The heavy chain constant regions thatcorrespond to the different classes of antibodies are called a, 6, ε, γ,and μ, respectively. The light chain constant regions (CL) which can befound in all five antibody classes are called κ (kappa) and λ (lambda).

The term “constant region derived from human origin” as used in thecurrent application denotes a constant heavy chain region of a humanantibody of the subclass IgG1, IgG2, IgG3, or IgG4 and/or a constantlight chain kappa or lambda region. Such constant regions are well knownin the state of the art and e.g. described by Kabat, E. A., (see e.g.Johnson, G. and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218; Kabat,E. A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788).

While antibodies of the IgG4 subclass show reduced Fc receptor(FcγRIIIa) binding, antibodies of other IgG subclasses show strongbinding. However Pro238, Asp265, Asp270, Asn297 (loss of Fccarbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254,Lys288, Thr307, Gln311, Asn434, and His435 are residues which, ifaltered, provide also reduced Fc receptor binding (Shields, R. L., etal., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al., FASEB J. 9(1995) 115-119; Morgan, A., et al, Immunology 86 (1995) 319-324; EP 0307 434).

In one embodiment an antigen binding protein according to the inventionhas a reduced FcR binding compared to an IgG1 antibody and the fulllength parent antibody is in regard to FcR binding of IgG4 subclass orof IgG1 or IgG2 subclass with a mutation in 5228, L234, L235 and/orD265, and/or contains the PVA236 mutation. In one embodiment themutations in the full length parent antibody are S228P, L234A, L235A,L235E and/or PVA236. In another embodiment the mutations in the fulllength parent antibody are in IgG4 S228P and in IgG1 L234A and L235A.

The constant region of an antibody is directly involved in ADCC(antibody-dependent cell-mediated cytotoxicity) and CDC(complement-dependent cytotoxicity). Complement activation (CDC) isinitiated by binding of complement factor C1q to the constant region ofmost IgG antibody subclasses. Binding of C1q to an antibody is caused bydefined protein-protein interactions at the so called binding site. Suchconstant region binding sites are known in the state of the art anddescribed e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981)2555-2560; Brunhouse, R. and Cebra, J. J., Mol. Immunol. 16 (1979)907-917; Burton, D. R., et al., Nature 288 (1980) 338-344; Thommesen,J., E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E., E., etal., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75(2001) 12161-12168; Morgan, A., et al, Immunology 86 (1995) 319-324; andEP 0 307 434. Such constant region binding sites are, e.g.,characterized by the amino acids L234, L235, D270, N297, E318, K320,K322, P331, and P329 (numbering according to EU index of Kabat).

The term “antibody-dependent cellular cytotoxicity (ADCC)” refers tolysis of human target cells by an antigen binding protein according tothe invention in the presence of effector cells. ADCC is measuredpreferably by the treatment of a preparation of antigen expressing cellswith an antigen binding protein according to the invention in thepresence of effector cells such as freshly isolated PBMC or purifiedeffector cells from buffy coats, like monocytes or natural killer (NK)cells or a permanently growing NK cell line.

The term “complement-dependent cytotoxicity (CDC)” denotes a processinitiated by binding of complement factor C1q to the Fc part of most IgGantibody subclasses. Binding of C1q to an antibody is caused by definedprotein-protein interactions at the so called binding site. Such Fc partbinding sites are known in the state of the art (see above). Such Fcpart binding sites are, e.g., characterized by the amino acids L234,L235, D270, N297, E318, K320, K322, P331, and P329 (numbering accordingto EU index of Kabat). Antibodies of subclass IgG1, IgG2, and IgG3usually show complement activation including C1q and C3 binding, whereasIgG4 does not activate the complement system and does not bind C1qand/or C3.

Cell-mediated effector functions of monoclonal antibodies can beenhanced by engineering their oligosaccharide component as described inUmana, P., et al., Nature Biotechnol. 17 (1999) 176-180, and U.S. Pat.No. 6,602,684. IgG1 type antibodies, the most commonly used therapeuticantibodies, are glycoproteins that have a conserved N-linkedglycosylation site at Asn297 in each CH2 domain. The two complexbiantennary oligosaccharides attached to Asn297 are buried between theCH2 domains, forming extensive contacts with the polypeptide backbone,and their presence is essential for the antibody to mediate effectorfunctions such as antibody dependent cellular cytotoxicity (ADCC)(Lifely, M., R., et al., Glycobiology 5 (1995) 813-822; Jefferis, R., etal., Immunol. Rev. 163 (1998) 59-76; Wright, A., and Morrison, S., L.,Trends Biotechnol. 15 (1997) 26-32). Umana, P., et al. NatureBiotechnol. 17 (1999) 176-180 and WO 99/54342 showed that overexpressionin Chinese hamster ovary (CHO) cells ofß(1,4)-N-acetylglucosaminyltransferase III (“GnTIII”), aglycosyltransferase catalyzing the formation of bisectedoligosaccharides, significantly increases the in vitro ADCC activity ofantibodies. Alterations in the composition of the Asn297 carbohydrate orits elimination affect also binding to FcγR and C1q (Umana, P., et al.,Nature Biotechnol. 17 (1999) 176-180; Davies, J., et al., Biotechnol.Bioeng. 74 (2001) 288-294; Mimura, Y., et al., J. Biol. Chem. 276 (2001)45539-45547; Radaev, S., et al., J. Biol. Chem. 276 (2001) 16478-16483;Shields, R., L., et al., J. Biol. Chem. 276 (2001) 6591-6604; Shields,R., L., et al., J. Biol. Chem. 277 (2002) 26733-26740; Simmons, L., C.,et al., J. Immunol. Methods 263 (2002) 133-147).

Methods to enhance cell-mediated effector functions of monoclonalantibodies are reported e.g. in WO 2005/018572, WO 2006/116260, WO2006/114700, WO 2004/065540, WO 2005/011735, WO 2005/027966, WO1997/028267, US 2006/0134709, US 2005/0054048, US 2005/0152894, WO2003/035835, WO 2000/061739.

In one preferred embodiment of the invention, the bispecific antigenbinding protein is glycosylated (if it comprises an Fc part of IgG1,IgG2, IgG3 or IgG4 subclass, preferably of IgG1 or IgG3 subclass) with asugar chain at Asn297 whereby the amount of fucose within the sugarchain is 65% or lower (Numbering according to Kabat). In anotherembodiment is the amount of fucose within the sugar chain is between 5%and 65%, preferably between 20% and 40%. “Asn297” according to theinvention means amino acid asparagine located at about position 297 inthe Fc region. Based on minor sequence variations of antibodies, Asn297can also be located some amino acids (usually not more than ±3 aminoacids) upstream or downstream of position 297, i.e. between position 294and 300. In one embodiment the glycosylated antigen binding proteinaccording to the invention the IgG subclass is of human IgG1 subclass,of human IgG1 subclass with the mutations L234A and L235A or of IgG3subclass. In a further embodiment the amount of N-glycolylneuraminicacid (NGNA) is 1% or less and/or the amount of N-terminalalpha-1,3-galactose is 1% or less within the sugar chain. The sugarchain show preferably the characteristics of N-linked glycans attachedto Asn297 of an antibody recombinantly expressed in a CHO cell.

The term “the sugar chains show characteristics of N-linked glycansattached to Asn297 of an antibody recombinantly expressed in a CHO cell”denotes that the sugar chain at Asn297 of the full length parentantibody according to the invention has the same structure and sugarresidue sequence except for the fucose residue as those of the sameantibody expressed in unmodified CHO cells, e.g. as those reported in WO2006/103100.

The term “NGNA” as used within this application denotes the sugarresidue N-glycolylneuraminic acid.

Glycosylation of human IgG1 or IgG3 occurs at Asn297 as core fucosylatedbiantennary complex oligosaccharide glycosylation terminated with up totwo Gal residues. Human constant heavy chain regions of the IgG1 or IgG3subclass are reported in detail by Kabat, E., A., et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), and by Brueggemann,M., et al., J. Exp. Med. 166 (1987) 1351-1361; Love, T., W., et al.,Methods Enzymol. 178 (1989) 515-527. These structures are designated asG0, G1 (α-1,6- or α-1,3-), or G2 glycan residues, depending from theamount of terminal Gal residues (Raju, T., S., Bioprocess Int. 1 (2003)44-53). CHO type glycosylation of antibody Fc parts is e.g. described byRoutier, F., H., Glycoconjugate J. 14 (1997) 201-207. Antibodies whichare recombinantly expressed in non-glycomodified CHO host cells usuallyare fucosylated at Asn297 in an amount of at least 85%. The modifiedoligosaccharides of the full length parent antibody may be hybrid orcomplex. Preferably the bisected, reduced/not-fucosylatedoligosaccharides are hybrid. In another embodiment, the bisected,reduced/not-fucosylated oligosaccharides are complex.

According to the invention “amount of fucose” means the amount of thesugar within the sugar chain at Asn297, related to the sum of allglycostructures attached to Asn297 (e.g. complex, hybrid and highmannose structures) measured by MALDI-TOF mass spectrometry andcalculated as average value. The relative amount of fucose is thepercentage of fucose-containing structures related to allglycostructures identified in an N-Glycosidase F treated sample (e.g.complex, hybrid and oligo- and high-mannose structures, resp.) byMALDI-TOF.

The antigen binding protein according to the invention is produced byrecombinant means. Thus, one aspect of the current invention is anucleic acid encoding the antigen binding protein according to theinvention and a further aspect is a cell comprising the nucleic acidencoding an antigen binding protein according to the invention. Methodsfor recombinant production are widely known in the state of the art andcomprise protein expression in prokaryotic and eukaryotic cells withsubsequent isolation of the antigen binding protein and usuallypurification to a pharmaceutically acceptable purity. For the expressionof the antibodies as aforementioned in a host cell, nucleic acidsencoding the respective modified light and heavy chains are insertedinto expression vectors by standard methods. Expression is performed inappropriate prokaryotic or eukaryotic host cells like CHO cells, NS0cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells, and the antigen binding protein is recovered from the cells(supernatant or cells after lysis). General methods for recombinantproduction of antibodies are well-known in the state of the art anddescribed, for example, in the review articles of Makrides, S. C.,Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-160; Werner, R. G., Drug Res. 48 (1998) 870-880.

The bispecific antigen binding proteins according to the invention aresuitably separated from the culture medium by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography. DNA or RNA encoding the monoclonalantibodies is readily isolated and sequenced using conventionalprocedures. The hybridoma cells can serve as a source of such DNA andRNA. Once isolated, the DNA may be inserted into expression vectors,which are then transfected into host cells such as HEK 293 cells, CHOcells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of recombinant monoclonal antibodies inthe host cells.

Amino acid sequence variants (or mutants) of the bispecific antigenbinding protein are prepared by introducing appropriate nucleotidechanges into the antigen binding protein DNA, or by nucleotidesynthesis. Such modifications can be performed, however, only in a verylimited range, e.g. as described above. For example, the modificationsdo not alter the above mentioned antibody characteristics such as theIgG isotype and antigen binding, but may improve the yield of therecombinant production, protein stability or facilitate thepurification.

The term “host cell” as used in the current application denotes any kindof cellular system which can be engineered to generate the antibodiesaccording to the current invention. In one embodiment HEK293 cells andCHO cells are used as host cells. As used herein, the expressions“cell,” “cell line,” and “cell culture” are used interchangeably and allsuch designations include progeny. Thus, the words “transformants” and“transformed cells” include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological activity as screened for inthe originally transformed cell are included.

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

A nucleic acid is “operably linked” when it is placed in a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Purification of antibodies is performed in order to eliminate cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis, and otherswell known in the art. See Ausubel, F., et al., ed. Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987). Different methods are well established and widespread used forprotein purification, such as affinity chromatography with microbialproteins (e.g. protein A or protein G affinity chromatography), ionexchange chromatography (e.g. cation exchange (carboxymethyl resins),anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75(1998) 93-102).

One aspect of the invention is a pharmaceutical composition comprisingan antigen binding protein according to the invention. Another aspect ofthe invention is the use of an antigen binding protein according to theinvention for the manufacture of a pharmaceutical composition. A furtheraspect of the invention is a method for the manufacture of apharmaceutical composition comprising an antigen binding proteinaccording to the invention. In another aspect, the present inventionprovides a composition, e.g. a pharmaceutical composition, containing anantigen binding protein according to the present invention, formulatedtogether with a pharmaceutical carrier.

Another aspect of the invention is the pharmaceutical composition forthe treatment of cancer.

Another aspect of the invention is the bispecific antigen bindingprotein according to the invention for the treatment of cancer.

Another aspect of the invention is the use of an antigen binding proteinaccording to the invention for the manufacture of a medicament for thetreatment of cancer.

Another aspect of the invention is a method of treatment of a patientsuffering from cancer by administering an antigen binding proteinaccording to the invention to the patient in the need of such treatment.

As used herein, “pharmaceutical carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion).

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. To administer a compound of the invention bycertain routes of administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation. For example, the compound may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intra-arterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The term cancer as used herein refers to proliferative diseases, such aslymphomas, lymphocytic leukemias, lung cancer, non small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma and Ewings sarcoma, including refractory versions ofany of the above cancers, or a combination of one or more of the abovecancers.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carrierpreferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham, F., L., and Van der Eb, A., J., Virology 52 (1973) 456-467.However, other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used. If prokaryotic cellsor cells which contain substantial cell wall constructions are used,e.g. one method of transfection is calcium treatment using calciumchloride as described by Cohen, S., N., et al, PNAS. 69 (1972)2110-2114.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as transcript) is subsequently beingtranslated into peptides, polypeptides, or proteins. The transcripts andthe encoded polypeptides are collectively referred to as gene product.If the polynucleotide is derived from genomic DNA, expression in aeukaryotic cell may include splicing of the mRNA.

A “vector” is a nucleic acid molecule, in particular self-replicating,which transfers an inserted nucleic acid molecule into and/or betweenhost cells. The term includes vectors that function primarily forinsertion of DNA or RNA into a cell (e.g., chromosomal integration),replication of vectors that function primarily for the replication ofDNA or RNA, and expression vectors that function for transcriptionand/or translation of the DNA or RNA. Also included are vectors thatprovide more than one of the functions as described.

An “expression vector” is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into apolypeptide. An “expression system” usually refers to a suitable hostcell comprised of an expression vector that can function to yield adesired expression product.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

DESCRIPTION OF THE SEQUENCE LISTING

-   SEQ ID NO:1 unmodified heavy chain <Ang-2> with C-terminal fused    <VEGF> VH-CL domains of modified Fab Fragment (CH1-CL exchange)-   SEQ ID NO:2 <VEGF> VL-CH1 domains of modified Fab Fragment (CH1-CL    exchange)-   SEQ ID NO:3 unmodified light chain <Ang-2>-   SEQ ID NO:4 unmodified heavy chain <Ang-2> with N-terminal fused    <VEGF> VH-CL domains of modified Fab Fragment (CH1-CL exchange)-   SEQ ID NO:5 modified heavy chain <VEGF> (CH1-CL exchange) with    C-terminal fused <Ang-2> VH-CH1 domains of unmodified Fab Fragment

EXAMPLES Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)Amino acids of antibody chains are numbered and referred to according toEU numbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63(1969) 78-85; Kabat, E. A., et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md., (1991)).

Recombinant DNA Techniques

Standard methods are used to manipulate DNA as described in Sambrook, J.et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents are used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments are prepared from oligonucleotides made bychemical synthesis. The gene segments, which are flanked by singularrestriction endonuclease cleavage sites, are assembled by annealing andligation of oligonucleotides including PCR amplification andsubsequently cloned via the indicated restriction sites e.g. KpnI/SacIor AscI/PacI into a pPCRScript (Stratagene) based pGA4 cloning vector.The DNA sequences of the subcloned gene fragments are confirmed by DNAsequencing. Gene synthesis fragments are ordered according to givenspecifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences are determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 is usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described bispecific tetravalent antibodiesvariants of expression plasmids for transient expression (e.g. in HEK293EBNA or HEK293-F) cells based either on a cDNA organization with orwithout a CMV-Intron A promoter or on a genomic organization with a CMVpromoter are applied.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this        plasmid in E. coli, and    -   a ß-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the antibody gene is composed of the followingelements:

-   -   unique restriction site(s) at the 5′ end    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   followed by the Intron A sequence in the case of the cDNA        organization,    -   a 5′-untranslated region of a human antibody gene,    -   a immunoglobulin heavy chain signal sequence,    -   the human bispecific tetravalent antibody chain (wildtype or        with domain exchange) either as cDNA or as genomic organization        with an the immunoglobulin exon-intron organization    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

The fusion genes comprising the described antibody chains as describedbelow are generated by PCR and/or gene synthesis and assembled withknown recombinant methods and techniques by connection of the accordingnucleic acid segments e.g. using unique restriction sites in therespective vectors. The subcloned nucleic acid sequences are verified byDNA sequencing. For transient transfections larger quantities of theplasmids are prepared by plasmid preparation from transformed E. colicultures (Nucleobond A X, Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques are used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Bispecific tetravalent antibodies are expressed by transientco-transfection of the respective three expression plasmids inadherently growing HEK293-EBNA or in HEK29-F cells growing in suspensionas described below.

Transient Transfections in HEK293-EBNA System

Bispecific tetravalent antibodies are expressed by transientco-transfection of the respective three expression plasmids (e.g.encoding the modified heavy chain, as well as the corresponding lightand modified light chain) in adherently growing HEK293-EBNA cells (humanembryonic kidney cell line 293 expressing Epstein-Barr-Virus nuclearantigen; American type culture collection deposit number ATCC #CRL-10852, Lot. 959 218) cultivated in DMEM (Dulbecco's modified Eagle'smedium, Gibco) supplemented with 10% Ultra Low IgG FCS (fetal calfserum, Gibco), 2 mM L-Glutamine (Gibco), and 250 μg/mL Geneticin(Gibco). For transfection FuGENE™ 6 Transfection Reagent (RocheMolecular Biochemicals) is used in a ratio of FuGENE™ reagent (μl) toDNA (μg) of 4:1 (ranging from 3:1 to 6:1). Proteins are expressed fromthe respective plasmids using a molar ratio of (modified and wildtype)light chain and modified heavy chain encoding plasmids of 1:1:1(equimolar) ranging from 1:1:2 to 2:2:1, respectively. Cells are feededat day 3 with L-Glutamine ad 4 mM, Glucose [Sigma] and NAA [Gibco].Bispecific tetravalent antibody containing cell culture supernatants areharvested from day 5 to 11 after transfection by centrifugation andstored at −20° C. General information regarding the recombinantexpression of human immunoglobulins in e.g. HEK293 cells is given in:Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Transient Transfections in HEK293-F System

Alternatively, bispecific tetravalent antibodies are generated bytransient transfection of the respective plasmids (e.g. encoding themodified heavy chain, as well as the corresponding light and modifiedlight chain) using the HEK293-F system (Invitrogen) according to themanufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growingin suspension either in a shake flask or in a stirred fermenter in serumfree FreeStyle 293 expression medium (Invitrogen) are transfected with amix of the three expression plasmids as described above and 293fectin orfectin (Invitrogen). For 2 L shake flask (Corning) HEK293-F cells areseeded at a density of 1.0E*6 cells/mL in 600 mL and incubated at 120rpm, 8% CO2. The day after the cells are transfected at a cell densityof ca. 1.5E*6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM(Invitrogen) with 600 μg total plasmid DNA (1 μg/mL) encoding themodified heavy chain, the corresponding light chain and thecorresponding modified light chain in an equimolar ratio and B) 20 mLOpti-MEM+1.2 mL 293 fectin or fectin (2 μl/mL). According to the glucoseconsumption glucose solution is added during the course of thefermentation. The supernatant containing the secreted antibody isharvested after 5-10 days and antibodies are either directly purifiedfrom the supernatant or the supernatant is frozen and stored.

Protein Determination

The protein concentration of purified bispecific tetravalent antibodiesand derivatives is determined by determining the optical density (OD) at280 nm, using the molar extinction coefficient calculated on the basisof the amino acid sequence according to Pace, C., N., et al., ProteinScience 4 (1995) 2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of bispecific tetravalent antibodies in cell culturesupernatants is estimated by immunoprecipitation with Protein AAgarose-beads (Roche). 60 μL Protein A Agarose beads are washed threetimes in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40).Subsequently, 1-15 mL cell culture supernatant are applied to theProtein A Agarose beads pre-equilibrated in TBS-NP40. After incubationfor at 1 h at room temperature the beads are washed on anUltrafree-MC-filter column (Amicon] once with 0.5 mL TBS-NP40, twicewith 0.5 mL 2× phosphate buffered saline (2×PBS, Roche) and briefly fourtimes with 0.5 mL 100 mM Na-citrate pH 5,0. Bound antibody is eluted byaddition of 35 μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of thesample is combined with NuPAGE® Sample Reducing Agent or left unreduced,respectively, and heated for 10 min at 70° C. Consequently, 5-30 μl areapplied to an 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen) (with MOPSbuffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® Antioxidantrunning buffer additive (Invitrogen) for reduced SDS-PAGE) and stainedwith Coomassie Blue.

The concentration of bispecific tetravalent antibodies in cell culturesupernatants is quantitatively measured by affinity HPLC chromatography.Briefly, cell culture supernatants containing antibodies and derivativesthat bind to Protein A are applied to an Applied Biosystems Poros A/20column in 200 mM KH2PO4, 100 mM sodium citrate, pH 7.4 and eluted fromthe matrix with 200 mM NaCl, 100 mM citric acid, pH 2.5 on an AgilentHPLC 1100 system. The eluted protein is quantified by UV absorbance andintegration of peak areas. A purified standard IgG1 antibody served as astandard.

Alternatively, the concentration of bispecific tetravalent antibodies incell culture supernatants is measured by Sandwich-IgG-ELISA. Briefly,StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche)are coated with 100 μL/well biotinylated anti-human IgG capture moleculeF(ab′)2<h-Fcγ> BI (Dianova) at 0.1 μg/mL for 1 h at room temperature oralternatively over night at 4° C. and subsequently washed three timeswith 200 μL/well PBS, 0.05% Tween (PBST, Sigma). 100 μL/well of adilution series in PBS (Sigma) of the respective antibody containingcell culture supernatants is added to the wells and incubated for 1-2 hon a microtiterplate shaker at room temperature. The wells are washedthree times with 200 μL/well PBST and bound antibody is detected with100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as detection antibody for1-2 h on a microtiterplate shaker at room temperature. Unbound detectionantibody is washed away three times with 200 μL/well PBST and the bounddetection antibody is detected by addition of 100 μL ABTS/well.Determination of absorbance is performed on a Tecan Fluor Spectrometerat a measurement wavelength of 405 nm (reference wavelength 492 nm).

Protein Purification

Proteins are purified from filtered cell culture supernatants referringto standard protocols. In brief, bispecific tetravalent antibodies areapplied to a Protein A Sepharose column (GE healthcare) and washed withPBS. Elution of bispecific tetravalent antibodies is achieved at pH 2.8followed by immediate neutralization of the sample. Aggregated proteinis separated from monomeric antibodies by size exclusion chromatography(Superdex 200, GE Healthcare) in PBS or in 20 mM Histidine, 150 mM NaClpH 6.0. Monomeric fractions are pooled, concentrated if required usinge.g. a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozenand stored at −20° C. or −80° C. Part of the samples are provided forsubsequent protein analytics and analytical characterization e.g. bySDS-PAGE, size exclusion chromatography or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) is used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer is used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography for the determination of the aggregationand oligomeric state of bispecific tetravalent antibodies is performedby HPLC chromatography. Briefly, Protein A purified antibodies areapplied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mMKH2PO4/K2HPO4, pH 7.5 on an Agilent HPLC 1100 system or to a Superdex200 column (GE Healthcare) in 2×PBS on a Dionex HPLC-System. The elutedprotein is quantified by UV absorbance and integration of peak areas.BioRad Gel Filtration Standard 151-1901 served as a standard.

Mass Spectrometry

The total deglycosylated mass of the bispecific tetravalent antibodiesis determined and confirmed via electrospray ionization massspectrometry (ESI-MS). Briefly, 100 μg purified antibodies aredeglycosylated with 50 mU N-Glycosidase F (PNGaseF, ProZyme) in 100 mMKH2PO4/K2HPO4, pH 7 at 37° C. for 12-24 h at a protein concentration ofup to 2 mg/mL and subsequently desalted via HPLC on a Sephadex G25column (GE Healthcare). The mass of the respective modified heavy, lightchain and modified light chain is determined by ESI-MS afterdeglycosylation and reduction. In brief, 50 μg bispecific tetravalentantibody in 115 μl are incubated with 60 μl 1M TCEP and 50 μl 8 MGuanidine-hydrochloride subsequently desalted. The total mass and themass of the reduced heavy and light chains is determined via ESI-MS on aQ-Star Elite MS system equipped with a NanoMate source.

VEGF Binding ELISA

The binding properties of the bispecific tetravalent antibodies isevaluated in an ELISA assay with full-length VEGF165-His protein (R&DSystems). For this sake Falcon polystyrene clear enhanced microtiterplates are coated with 100 μl 2 μg/mL recombinant human VEGF165 (R&DSystems) in PBS for 2 h at room temperature or over night at 4° C. Thewells are washed three times with 300 μl PBST (0.2% Tween 20) andblocked with 200 μl 2% BSA 0.1% Tween 20 for 30 min at room temperatureand subsequently washed three times with 300 μl PBST. 100 μL/well of adilution series of the purified bispecific tetravalent antibodies in PBS(Sigma) is added to the wells and incubated for 1 h on a microtiterplateshaker at room temperature. The wells are washed three times with 300 μlPBST (0.2% Tween 20) and bound antibody is detected with 100 μL/well 0.1μg/mL F(ab′) <hFcgamma>POD (Immuno research) in 2% BSA 0.1% Tween 20 asdetection antibody for 1 h on a microtiterplate shaker at roomtemperature. Unbound detection antibody is washed away three times with300 μL/well PBST and the bound detection antibody is detected byaddition of 100 μL ABTS/well. Determination of absorbance is performedon a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm(reference wavelength 492 nm).

VEGF Binding: Kinetic Characterization of VEGF Binding at 37° C. bySurface Plasmon Resonance (Biacore)

In order to further corroborate the ELISA findings the binding of thebispecific tetravalent antibodies to VEGF is quantitatively analyzedusing surface plasmon resonance technology on a Biacore T100 instrumentaccording to the following protocol and analyzed using the T100 softwarepackage: Briefly, bispecific tetravalent antibodies are captured on aCMS-Chip via binding to a Goat Anti Human IgG (JIR 109-005-098). Thecapture antibody is immobilized by amino coupling using standard aminocoupling as follows: HBS-N buffer served as running buffer, activationis done by mixture of EDC/NHS with the aim for a ligand density of 700RU. The Capture-Antibody is diluted in coupling buffer NaAc, pH 5.0, c=2μg/mL, finally still activated carboxyl groups are blocked by injectionof 1 M Ethanolamine. Capturing of bispecific tetravalent <VEGF>antibodies is done at a flow of 5 μL/min and c=10 nM, diluted withrunning buffer+1 mg/mL BSA; a capture level of approx. 30 RU should bereached. rhVEGF (rhVEGF, R&D-Systems Cat.-No, 293-VE) is used asanalyte. The kinetic characterization of VEGF binding to bispecifictetravalent <VEGF> antibodies is performed at 25° C. or 37° C. inPBS+0.005% (v/v) Tween20 as running buffer. The sample is injected witha flow of 50 μL/min and an association of time 80 sec. and adissociation time of 1200 sec with a concentration series of rhVEGF from300-0.29 nM. Regeneration of free capture antibody surface is performedwith 10 mM Glycin pH 1.5 and a contact time of 60 sec after each analytecycle. Kinetic constants are calculated by using the usual doublereferencing method (control reference: binding of rhVEGF to capturemolecule Goat Anti Human IgG, blanks on the measuring flow cell, rhVEGFconcentration “0”, Model: Langmuir binding 1:1, (Rmax set to localbecause of capture molecule binding).

Ang-2 Binding ELISA

The binding properties of the bispecific tetravalent antibodies isevaluated in an ELISA assay with full-length Ang-2-His protein (R&DSystems). For this sake Falcon polystyrene clear enhanced microtiterplates are coated with 100 μl 1 μg/mL recombinant human Ang-2 (R&DSystems, carrier-free) in PBS for 2 h at room temperature or over nightat 4° C. The wells are washed three times with 300 μl PBST (0.2% Tween20) and blocked with 200 μl 2% BSA 0.1% Tween 20 for 30 min at roomtemperature and subsequently washed three times with 300 μl PBST. 100μL/well of a dilution series of the purified bispecific tetravalentantibodies in PBS (Sigma) is added to the wells and incubated for 1 h ona microtiterplate shaker at room temperature. The wells are washed threetimes with 300 μl PBST (0.2% Tween 20) and bound antibody is detectedwith 100 μL/well 0.1 μg/mL F(ab′) <hk>POD (Biozol Cat. No. 206005) in 2%BSA 0.1% Tween 20 as detection antibody for 1 h on a microtiterplateshaker at room temperature. Unbound detection antibody is washed awaythree times with 300 μL/well PBST and the bound detection antibody isdetected by addition of 100 μL ABTS/well. Determination of absorbance isperformed on a Tecan Fluor Spectrometer at a measurement wavelength of405 nm (reference wavelength 492 nm).

Ang-2 Binding BIACORE

Binding of the bispecific tetravalent antibodies to human Ang-2 isinvestigated by surface plasmon resonance using a BIACORE T100instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, foraffinity measurements goat<hIgG-Fcγ> polyclonal antibodies areimmobilized on a CMS chip via amine coupling for presentation of thebispecific tetravalent antibodies against human Ang-2. Binding ismeasured in HBS buffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% Tween20, ph 7.4), 25° C. Purified Ang-2-His (R&D systems or in housepurified) is added in various concentrations in solution. Association ismeasured by an Ang-2-injection of 3 minutes; dissociation is measured bywashing the chip surface with HBS buffer for 3 minutes and a KD value isestimated using a 1:1 Langmuir binding model. Due to heterogenity of theAng-2 preparation no 1:1 binding can be observed; KD values are thusonly relative estimations. Negative control data (e.g. buffer curves)are subtracted from sample curves for correction of system intrinsicbaseline drift and for noise signal reduction. Biacore T100 EvaluationSoftware version 1.1.1 is used for analysis of sensorgrams and forcalculation of affinity data. Alternatively, Ang-2 could be capturedwith a capture level of 2000-1700 RU via a PentaHisAntibody (PentaHis-AbBSA-free, Qiagen No. 34660) that is immobilized on a CMS chip via aminecoupling (BSA-free) (see below).

Ang-2-VEGF Bridging ELISA

The binding properties of the bispecific tetravalent antibodies isevaluated in an ELISA assay with immobilized full-length VEGF165-Hisprotein (R&D Systems) and human Ang-2-His protein (R&D Systems) fordetection of bound bispecific antibody. Only a bispecific tetravalent<VEGF-Ang-2> antibody is able to o bind simultaneously to VEGF and Ang-2and thus bridge the two antigens whereas monospecific “standard” IgG1antibodies is not be capable of simultaneously binding to VEGF and Ang-2(FIG. 7).

For this sake Falcon polystyrene clear enhanced microtiter plates arecoated with 100 μl 2 μg/mL recombinant human VEGF165 (R&D Systems) inPBS for 2 h at room temperature or over night at 4° C. The wells arewashed three times with 300 μl PBST (0.2% Tween 20) and blocked with 200μl 2% BSA 0.1% Tween 20 for 30 min at room temperature and subsequentlywashed three times with 300 μl PBST. 100 μL/well of a dilution series ofpurified bispecific tetravalent antibodies in PBS (Sigma) is added tothe wells and incubated for 1 h on a microtiterplate shaker at roomtemperature. The wells are washed three times with 300 μl PBST (0.2%Tween 20) and bound antibody is detected by addition of 100 μl 0.5 μg/mLhuman Ang-2-His (R&D Systems) in PBS. The wells are washed three timeswith 300 μl PBST (0.2% Tween 20) and bound Ang-2 is detected with 100 μl0.5 μg/mL<Ang-2>mIgG1-Biotin antibody (BAM0981, R&D Systems) for 1 h atroom temperature. Unbound detection antibody is washed away with threetimes 300 μl PBST (0.2% Tween 20) and bound antibody is detected byaddition of 100 μl 1:2000 Streptavidin-POD conjugate (Roche DiagnosticsGmbH, Cat. No. 11089153) 1:4 diluted in blocking buffer for 1 h at roomtemperature. Unbound Streptavidin-POD conjugate is washed away withthree-six times 300 μl PBST (0.2% Tween 20) and bound Strepatavidin-PODconjugate is detected by addition of 100 μL ABTS/well. Determination ofabsorbance is performed on a Tecan Fluor Spectrometer at a measurementwavelength of 405 nm (reference wavelength 492 nm).

Demonstration of Simultaneous Binding of Bispecific Tetravalent Antibody<VEGF-Ang-2> to VEGF-A and Ang-2 by Biacore

In order to further corroborate the data from the bridging ELISA anadditional assay is established to confirm simultaneous binding to VEGFand Ang-2 using surface plasmon resonance technology on a Biacore T100instrument according to the following protocol and analyzed using theT100 software package (T100 Control, Version 2.01, T100 Evaluation,Version 2.01, T100 Kinetics Summary, Version 1.01): Ang-2 is capturedwith a capture level of 2000-1700 RU in PBS, 0.005% (v/v) Tween20running buffer via a PentaHisAntibody (PentaHis-Ab BSA-free, Qiagen No.34660) that is immobilized on a CMS chip via amine coupling (BSA-free).HBS-N buffer served as running buffer during coupling, activation isdone by mixture of EDC/NHS. The PentaHis-Ab BSA-free Capture-Antibody isdiluted in coupling buffer NaAc, pH 4.5, c=30 μg/mL, finally stillactivated carboxyl groups are blocked by injection of 1 M Ethanolamine;ligand densities of 5000 and 17000 RU are tested. Ang-2 with aconcentration of 500 nM is captured by the PentaHis-Ab at a flow of 5μL/min diluted with running buffer+1 mg/mL BSA. Subsequently, <Ang-2,VEGF> bispecific tetravalent antibody binding to Ang-2 and to VEGF isdemonstrated by incubation with rhVEGF and formation of a sandwichcomplex. For this sake, the bispecific tetravalent <VEGF-Ang-2> antibodyis bound to Ang-2 at a flow of 50 μL/min and a concentration of 100 nM,diluted with running buffer+1 mg/mL BSA and simultaneous binding isdetected by incubation with VEGF (rhVEGF, R&D-Systems Cat.-No, 293-VE)in PBS+0.005% (v/v) Tween20 running buffer at a flow of 50 μL/min and aVEGF concentration of 150 nM. Association time 120 sec, dissociationtime 1200 sec. Regeneration is done after each cycle at a flow of 50μL/min with 2×10 mM Glycin pH 2.0 and a contact time of 60 sec.Sensorgrams are corrected using the usual double referencing (controlreference: binding of bispecific antibody and rhVEGF to capture moleculePentaHisAb). Blanks for each Ab are measured with rhVEGF concentration“0”.

Generation of HEK293-Tie2 Cell Line

In order to determine the interference of <Ang-2, VEGF> bispecifictetravalent antibodies with Ang-2 stimulated Tie2 phosphorylation andbinding of Ang-2 to Tie2 on cells a recombinant HEK293-Tie cell line wasgenerated. Briefly, a pcDNA3 based plasmid (RB22-pcDNA3 Topo hTie2)coding for full-length human Tie2 under control of a CMV promoter and aNeomycin resistance marker was transfected using Fugene (Roche AppliedScience) as transfection reagent into HEK293 cells (ATCC) and resistantcells were selected in DMEM 10% FCS, 500 μg/mL G418. Individual cloneswere isolated via a cloning cylinder, and subsequently analyzed for Tie2expression by FACS. Clone 22 was identified as clone with high andstable Tie2 expression even in the absence of G418 (HEK293-Tie2clone22). HEK293-Tie2 clone22 is subsequently used for cellular assays:Ang-2 induced Tie2 phosphorylation and Ang-2 cellular ligand bindingassay.

Ang-2 Induced Tie2 Phosphorylation Assay

Inhibition of Ang-2 induced Tie2 phosphorylation by <Ang-2, VEGF>bispecific tetravalent antibodies is measured according to the followingassay principle. HEK293-Tie2 clone22 is stimulated with Ang-2 for 5minutes in the absence or presence of Ang-2 antibody and P-Tie2 isquantified by a sandwich ELISA. Briefly, 2×105 HEK293-Tie2 clone 22cells per well are grown over night on a Poly-D-Lysine coated 96well-microtiter plate in 100 μl DMEM, 10% FCS, 500 μg/mL Geneticin. Thenext day a titration row of <Ang-2, VEGF> bispecific tetravalentantibodies is prepared in a microtiter plate (4-fold concentrated, 75 μlfinal volume/well, duplicates) and mixed with 75 μl of an Ang-2 (R&Dsystems #623-AN] dilution (3.2 μg/mL as 4-fold concentrated solution).Antibodies and Ang-2 are pre-incubated for 15 min at room temperature.100 μl of the mix are added to the HEK293-Tie2 clone 22 cells(pre-incubated for 5 min with 1 mM NaV3O4, Sigma #S6508) and incubatedfor 5 min at 37° C. Subsequently, cells are washed with 200 μl ice-coldPBS+1 mM NaV3O4 per well and lysed by addition of 120 μl lysis buffer(20 mM Tris, pH 8.0, 137 mM NaCl, 1% NP-40, 10% glycerol, 2 mM EDTA, 1mM NaV3O4, 1 mM PMSF and 10 μg/mL Aprotinin) per well on ice. Cells arelysed for 30 min at 4° C. on a microtiter plate shaker and 100 μl lysateare transferred directly into a p-Tie2 ELISA microtiter plate (R&DSystems, R&D #DY990) without previous centrifugation and without totalprotein determination. P-Tie2 amounts are quantified according to themanufacturer's instructions and IC50 values for inhibition aredetermined using XLfit4 analysis plug-in for Excel (Dose-response onesite, model 205). IC50 values can be compared within on experiment butmight vary from experiment to experiment.

VEGF Induced HUVEC Proliferation Assay

VEGF induced HUVEC (Human Umbilical Vein Endothelial Cells, Promocell#C-12200) proliferation is chosen to measure the cellular function of<Ang-2, VEGF> bispecific tetravalent antibodies. Briefly, 5000 HUVECcells (low passage number, ≤5 passages) per 96 well are incubated in 100μl starvation medium (EBM-2 Endothelial basal medium 2, Promocell #C-22211, 0.5% FCS, Penicilline/Streptomycine) in a collagen I-coated BDBiocoat Collagen I 96-well microtiter plate (BD #354407/35640 overnight. Varying concentrations of <Ang-2, VEGF> bispecific tetravalentantibody are mixed with rhVEGF (30 ng1/mL final concentration, BD#354107) and pre-incubated for 15 minutes at room temperature.Subsequently, the mix is added to the HUVEC cells and they are incubatedfor 72 h at 37° C., 5% CO2. On the day of analysis the plate isequilibrated to room temperature for 30 min and cellviability/proliferation is determined using the CellTiter-Glo™Luminescent Cell Viability Assay kit according to the manual (Promega, #G7571/2/3). Luminescence is determined in a spectrophotometer.

Example 1 Production, Expression, Purification and Characterization of aBispecific and Tetravalent Antibody Recognizing Ang-2 and VEGF-A

In a first example a bispecific tetravalent antibody without a linkerbetween the respective antibody chains recognizing Ang-2 and VEGF-A wasmade by fusing via a (G4S)4-connector a VH-CL domain fusion againstVEGF-A to the C-terminus of the heavy chain of an antibody recognizingAng-2 (SEQ1 or a corresponding IgG1 allotype). In order to obtain thebispecific tetravalent antibody this heavy chain construct wasco-expressed with plasmids coding for the respective light chain of theAng-2 antibody (SEQ3) and a VL-CH1 domain fusion recognizing VEGF-A(SEQ2). The scheme of the respective antibody is given in FIG. 5.

The bispecific tetravalent antibody is generated was described in thegeneral methods section by classical molecular biology techniques and isexpressed transiently in HEK293F cells as described above. Subsequently,it was purified from the supernatant by a combination of Protein Aaffinity chromatography and size exclusion chromatography. The obtainedproduct was characterized for identity by mass spectrometry andanalytical properties such as purity by SDS-PAGE, monomer content andstability.

expression Titer purification [μg/mL] yield final product homogeneity(final product) 21 19.2 mg/L 95%

These data show that the bispecific tetravalent antibody can be producedin good yields and is stable.

Subsequently binding to Ang-2 and VEGF-A as well as simultaneous bindingwere studied by ELISA and Biacore assays described above and functionalproperties such as inhibition of Tie2 phosphorylation and inhibition ofVEGF induced HUVEC proliferation are analyzed showing that the generatedbispecific tetravalent antibody is able to bind to Ang-2 and VEGF-A andblock their activity simultaneously.

Example 2 Production, Expression, Purification and Characterization of aBispecific and Tetravalent Antibody Recognizing Ang-2 and VEGF-A

In a second example a bispecific tetravalent antibody without a linkerbetween the respective antibody chains recognizing Ang-2 and VEGF-A wasmade by fusing via a (G4S)4-connector a VH-CL domain fusion againstVEGF-A to the N-terminus of the heavy chain of an antibody recognizingAng-2 (SEQ4 or a corresponding IgG1 allotype). In order to obtain thebispecific tetravalent antibody this heavy chain construct wasco-expressed with plasmids coding for the respective light chain of theAng-2 antibody (SEQ3) and a VL-CH1 domain fusion recognizing VEGF-A(SEQ2). The scheme of the respective antibody is given in FIG. 6.

The bispecific tetravalent antibody was generated as described in thegeneral methods section by classical molecular biology techniques and isexpressed transiently in HEK293F cells as described above. Subsequently,it was purified from the supernatant by a combination of Protein Aaffinity chromatography and size exclusion chromatography. The obtainedproduct was characterized for identity by mass spectrometry andanalytical properties such as purity by SDS-PAGE, monomer content andstability.

expression Titer purification [μg/mL] yield final product homogeneity(final product) 18 12.4 mg/L 95%

These data show that the bispecific tetravalent antibody can be producedin good yields and is stable.

Subsequently binding to Ang-2 and VEGF-A as well as simultaneous bindingwere studied by ELISA and Biacore assays described above and functionalproperties such as inhibition of Tie2 phosphorylation and inhibition ofVEGF induced HUVEC proliferation are analyzed showing that the generatedbispecific tetravalent antibody is able to bind to Ang-2 and VEGF-A andblock their activity simultaneously.

Example 3 Production, Expression, Purification and Characterization of aBispecific and Tetravalent Antibody Recognizing Ang-2 and VEGF-A

In a third example a bispecific tetravalent antibody without a linkerbetween the respective antibody chains recognizing Ang-2 and VEGF-A wasmade by fusing via a (G4S)4-connector a VH-CH1 Fab domain against Ang-2to the C-terminus of the heavy chain of a CH1-CL exchange antibodyrecognizing VEGF (SEQ5 or a corresponding IgG1 allotype). In order toobtain the bispecific tetravalent antibody this heavy chain constructwas co-expressed with plasmids coding for the respective light chain ofthe Ang-2 antibody (SEQ3) and a VL-CH1 domain fusion recognizing VEGF-A(SEQ2). The scheme of the respective antibody is given in FIG. 7.

The bispecific tetravalent antibody was generated as described in thegeneral methods section by classical molecular biology techniques and isexpressed transiently in HEK293F cells as described above. Subsequently,it was purified from the supernatant by a combination of Protein Aaffinity chromatography and size exclusion chromatography. The obtainedproduct was characterized for identity by mass spectrometry andanalytical properties such as purity by SDS-PAGE, monomer content andstability.

expression Titer purification [μg/mL] yield final product homogeneity(final product) 8.5-9 1.8-4.1 mg/L 100%

These data show that the bispecific tetravalent antibody can be producedin good yields and is stable.

Subsequently binding to Ang-2 and VEGF-A as well as simultaneous bindingwere studied by ELISA and Biacore assays described above and functionalproperties such as inhibition of Tie2 phosphorylation and inhibition ofVEGF induced HUVEC proliferation are analyzed showing that the generatedbispecific tetravalent antibody is able to bind to Ang-2 and VEGF-A andblock their activity simultaneously.

1-10. (canceled) 11: A bispecific antigen binding protein, comprising:a) two light chains and two heavy chains of an antibody that comprisestwo Fab fragments and that specifically binds to a first antigen; and b)two additional Fab fragments of an antibody which specifically binds toa second antigen, wherein the additional Fab fragments are each fused atthe C- or N-termini of the heavy chains of a); wherein the bispecificantigen binding protein also comprises a structural modificationselected from the group consisting of: i) in both Fab fragments of a) orin both Fab fragments of b) the variable domains VL and VH are replacedby each other and the constant domains CL and CH1 are replaced by eachother, or the constant domains CL and CH1 are replaced by each other;ii) in both Fab fragments of a) the variable domains VL and VH arereplaced by each other, and the constant domains CL and CH1 are replacedby each other, and in both Fab fragments of b) the variable domains VLand VH are replaced by each other, or the constant domains CL and CH1are replaced by each other; iii) in both Fab fragments of a) thevariable domains VL and VH are replaced by each other, or the constantdomains CL and CH1 are replaced by each other, and in both Fab fragmentsof b) the variable domains VL and VH are replaced by each other, and theconstant domains CL and CH1 are replaced by each other; iv) in both Fabfragments of a) the variable domains VL and VH are replaced by eachother, and in both Fab fragments of b) the constant domains CL and CH1are replaced by each other; and v) in both Fab fragments of a) theconstant domains CL and CH1 are replaced by each other, and in both Fabfragments of b) the variable domains VL and VH are replaced by eachother. 12: The bispecific antigen binding protein according to claim 11,wherein in both Fab fragments of a) or in both Fab fragments of b) thevariable domains VL and VH are replaced by each other and the constantdomains CL and CH1 are replaced by each other. 13: The bispecificantigen binding protein according to claim 11, wherein in both Fabfragments of b) the variable domains VL and VH are replaced by eachother and the constant domains CL and CH1 are replaced by each other.14: The bispecific antigen binding protein according to claim 13,wherein each Fab fragment of b) is fused at the N-terminus of each ofthe heavy chains in a). 15: The bispecific antigen binding proteinaccording to claim 14, wherein the CL of each Fab fragment of b) isfused to the VH of each of the heavy chains in a). 16: The bispecificantigen binding protein according to claim 13, wherein the antibody ina) is a full length antibody. 17: The bispecific antigen binding proteinaccording to claim 14, wherein the antibody in a) is a full lengthantibody. 18: The bispecific antigen binding protein according to claim15 comprising a heavy chain comprising in N to C terminal directionVL-CL-VH-CH1-CH2-CH3, comprising a light chain comprising in N to Cterminal direction VH-CH, and comprising a light chain comprising in Nto C terminal direction VL-CL. 19: A pharmaceutical compositioncomprising the bispecific antigen binding protein according to claim 13and at least one pharmaceutically acceptable excipient.